Molluscan Compounds Provide Drug Leads for the Treatment and Prevention of Respiratory Disease

Respiratory diseases place an immense burden on global health and there is a compelling need for the discovery of new compounds for therapeutic development. Here, we identify research priorities by critically reviewing pre-clinical and clinical studies using extracts and compounds derived from molluscs, as well as traditional molluscan medicines, used in the treatment of respiratory diseases. We reviewed 97 biomedical articles demonstrating the anti-inflammatory, antimicrobial, anticancer, and immunomodulatory properties of >320 molluscan extracts/compounds with direct relevance to respiratory disease, in addition to others with promising bioactivities yet to be tested in the respiratory context. Of pertinent interest are compounds demonstrating biofilm inhibition/disruption and antiviral activity, as well as synergism with approved antimicrobial and chemotherapeutic agents. At least 100 traditional medicines, incorporating over 300 different mollusc species, have been used to treat respiratory-related illness in cultures worldwide for thousands of years. These medicines provide useful clues for the discovery of bioactive components that likely underpin their continued use. There is particular incentive for investigations into anti-inflammatory compounds, given the extensive application of molluscan traditional medicines for symptoms of inflammation, and shells, which are the principal molluscan product used in these preparations. Overall, there is a need to target research toward specific respiratory disease-related hypotheses, purify bioactive compounds and elucidate their chemical structures, and develop an evidence base for the integration of quality-controlled traditional medicines.


Respiratory Disease Pathology and Epidemiology
The role of the respiratory system is to allow continuous O 2 and CO 2 exchange with the environment [1]. In doing so, it incidentally permits exposure to airborne particles, chemicals, and infectious organisms with each breath [1,2]. As such, the nature and types of respiratory disease are many and physiological defense mechanisms must be well regulated. Also known as lung or pulmonary diseases, respiratory diseases are conditions affecting the lungs and other tissues of the respiratory system impairing normal gas exchange [3].
Respiratory diseases are among the leading causes of death and disability worldwide [2]. Asthma, chronic obstructive pulmonary disease (COPD), lung cancer, and communicable infections impose a  [2,6,27] Mar. Drugs 2020, 18, 570 4 of 50  [32] Abbreviations: DALYs: disability-adjusted life years-a metric that estimates the amount of active and productive life lost due to a condition; TB: tuberculosis; NTM: non-tuberculosis mycobacteria; RSV: respiratory syncytial virus; COVID-19: 2019 novel corona virus; CFTR: cystic fibrosis transmembrane conductance regulator; NO: nitric oxide. * Other important respiratory diseases not described in this table include sleep disordered breathing, pulmonary hypertension, and pulmonary embolism. † Typically caused by Streptococcus pneumoniae, Haemophilus influenzae type b, respiratory syncytial virus; Pneumocystis jiroveci (in HIV infected). ‡ Communicable refers to diseases caused by infectious agents that can be transmitted from one person (or animal) to another via. direct or indirect contact. Non-communicable diseases are not transmitted from one person (or animal) to another. § From December 2019 to time of publication. ¶ The annual mortality rate from respiratory infection is difficult to quantify, particularly in developing countries, and estimates vary considerably e.g., van der Poll & Opal (2009) estimate mortality rate of 4 million caused by pneumococcal pneumonia alone. # Particularly of Pseudemonas aeruginosa, Burkholdaria cepacia, Staphylococcus aureus, often multi-drug resistant.

Literature Search Methods and Evaluation
We undertook an extensive scoping review of published biomedical and ethnomedical literature using scientific databases Scopus, Web of Science, and PubMed. Databases were searched by article title, abstract, and keywords without date limits (as of October 2020). Full search criteria are provided as Supplementary Information. All records were screened by title and abstract before full-text articles were assessed.
Over 900 biomedical articles were screened based on their relevance to respiratory disease and focus on bioactivity of the molluscan extract or compound/s. In vitro studies were accepted if the test suite included at least one respiratory pathogenic microorganism or cell line of respiratory origin. Other non-respiratory microorganisms, cell types, and models that may also have been tested are not described herein, unless results were useful for comparison to respiratory microorganisms, cell types, or models. In vivo animal models and human clinical trials were accepted if the disease was specific to the respiratory system (e.g., asthma, infection, COPD, lung cancer) and the bioactivity of the molluscan extract/compound was evident in the results. Search results returned copious studies using Hc as a vaccine adjuvant or conjugate which were only accepted if treatments (or controls) used Hc alone or the antigen both with and without Hc. In vivo immunological studies using Hc as a model antigen were separated from those using Hc as a treatment or treatment component and accepted if the investigation was respiratory related. Key review articles [49,53,56,66,90,102,103] were searched for relevant references therein, which were then screened by abstract. Many of the studies included in previous reviews provide further insight into the bioactivity and immunogenicity of molluscan extracts/compounds and their potential to treat respiratory diseases, however are not specific to these diseases and therefore not included herein. In total, 97 peer-reviewed biomedical articles are included Mar. Drugs 2020, 18, 570 7 of 50 in this review. Those with substantial in vivo and in vitro components were regarded as separate studies in tables and analyses (total: 54 in vitro studies, 25 in vivo studies, 11 human clinical trials and 16 in vivo human/animal studies in which Hc was used as a model antigen).
For literature regarding traditional medicinal applications, database searches returned 113 results, minus duplicates. Key review papers by Ahmad et al. [49] and Benkendorff et al. [90] provided important references to books and monographs not recorded in databases. The Chinese Marine Materia Medica [104] was translated by the third author of this review. All texts (published in English or otherwise translated) were screened for mollusc-derived remedies relating to the treatment of respiratory disease or symptoms, and appear directly as translated with contemporary revisions provided in footnotes. In total, 22 traditional medicine studies/texts are included in this review. The phrase "other traditional medicines" used throughout refers to traditional medicines other than TCMs. We use the term traditional or molluscan "medicine" to refer to any concoction traditionally used to treat human ailments that contain molluscan products functioning as potential active pharmaceutical ingredients (APIs).
Data were collated and descriptive statistics calculated in Excel. Summary tables are provided throughout the review and as Supplementary Files. Table 2 summarises the number of different remedies and species traditionally used to treat specific symptoms of respiratory disease; full details of traditional medicines, including species, preparations, and indications are provided in Supplementary Tables S1 and S2. Table 3 provides a taxonomic breakdown of the number of species in each molluscan class as well as their division by terrestrial, freshwater, and marine habitat, that are used in traditional medicines and compares these to biomedical studies, whereas Table 4 provides the number of studies and species tested for different bioactivities. In both cases, the species, extract or compound, experimental design, effective concentrations and outcomes are detailed in Tables 5-8 to inform research direction and provide evidence that molluscan compounds could be the APIs in traditional medicines. Studies using molluscan Hcs as vaccine conjugates or model antigens are included in summary Tables 2  and 3 and detailed in Supplementary Tables S3 and S4. Selected patents and purified compounds that have been tested with relevance to respiratory disease are provided in Supplementary Tables S5 and S6, respectively. Taxonomic nomenclature of the mollusc species used throughout this review is corrected according to the World Register of Marine Species [105]. Where corrected, reference to species names used in the cited publications is provided in footnotes.

Traditional Molluscan Respiratory Medicines
Molluscs are a valued resource providing food, dye, shells, forming currencies and medicines, as well as being of symbolic and ritualistic importance to many historical and contemporary cultures around the world [90,[106][107][108]. Respiratory diseases have always concerned human health and sophisticated traditional medicine systems formed the basis of care long before the advent of modern pharmaceuticals. Many traditional respiratory medicines feature diverse preparations of the flesh, shell, opercula, eggs and secretions of different mollusc species, mostly sourced from local marine environments ( Table 2, Table 3 and Table S1, Figure 1). Records of molluscan respiratory medicines date back at least as far as Ancient Greece (800 BC), when the boiled flesh of Octopus vulgaris was used to relieve heavy nasal congestion with fever, treat infectious diseases, and strengthen the body's immune system [109] (Table S1). During the Middle Eastern Medieval Period (500-1500 AD), the sea hare Aplysia depilans was used in traditional Arabic medicine to alleviate dyspnea (difficult or labored breathing), dry cough, and hemoptysis (coughing up blood) (Table S1). Traditional Chinese Medicine (TCM) is considered one of the oldest (>3500 years old) and most well-documented systems of traditional medicine in the world [49]. Most TCMs are derived from plant/terrestrial natural products, although marine species are important with an entire volume dedicated to marine-derived remedies [104]. Of the marine TCMs, 111 are derived from molluscs and 61 of these are used to and S2; Figure 1). Flesh may be cooked and eaten [109,115] or applied externally [104,109] (Tables S1  and S2). In Ancient Greece, flesh of cephalopods (O. vulgaris, S. officianalis) and the giant trumpet shell (Charonia tritonis) were eaten for conditions of low immunity in addition to infectious or inflammatory respiratory diseases [109] (Table S1). Among Nigerian tribes, the flesh is "punctured to obtain the fluid" ( [123] pp. 491). These accounts could be related to circulating hemolymph (equivalent to blood), which represents at least 20% of molluscan body weight [137]. Hemolymph is composed largely of hemocyanin (Hc), which has a range of bioactivities and is among the most potent of immunogens used in respiratory disease-related studies, as discussed later in this review (Tables S3 and S4). Many other bioactive compounds (e.g., AMPs, proteins, peptides, and polyketides) have been derived from molluscan body tissue (Tables 5-8 Table S2), B) other traditional medicines (n = 39 remedies; Table S1) C) in vitro, in vivo and clinical biomedical studies (n = 97 articles; Tables 5-8 and S3; not  including studies using Hc as a model antigen Table S4). NA: not available, AM: adductor muscle, HBG: hypobranchial gland, DG: digestive gland, SG: salivary gland; shell includes cuttlebone; whole includes those listed as "whole animal" or "body and shell".

Supporting Evidence for the Bioactivity of Traditional Molluscan Respiratory Medicines
The bioactivity and therapeutic value of traditional molluscan medicines is for the most part speculative. However, there are growing bodies of substantiating evidence by virtue of biomedical studies using derivatives of hemolymph, mucus, body extracts, and specialized glands ( Figure 1, Tables 3-8). European 18-19th century physicians and pharmaceutical texts proclaimed the unsurpassed benefits of helicidine (mucus of the terrestrial snail H. pomatia) for alleviating various respiratory conditions such as whooping cough, TB, influenza, pneumonia, chronic bronchitis, and  Table S2), (B) other traditional medicines (n = 39 remedies; Table S1) (C) in vitro, in vivo and clinical biomedical studies (n = 97 articles; Tables 5-8 and Table S3; not including studies using Hc as a model antigen Table S4). NA: not available, AM: adductor muscle, HBG: hypobranchial gland, DG: digestive gland, SG: salivary gland; shell includes cuttlebone; whole includes those listed as "whole animal" or "body and shell".
Several recurrent themes emerge regarding the application of molluscan traditional medicines for certain respiratory diseases (Table 2). Many are indicated for the treatment of tuberculosis (TB), both pulmonary and extrapulmonary ( Table 2, Tables S1 and S2). The disease has been a permanent challenge over the course of human history, given the primordial origins of Mycobacterium tuberculosis [112] and it stands to reason that traditional treatments for TB infection (and the cough and inflammatory symptoms it causes) are ubiquitous. Alves et al. [113] documented the use of bivalve mollusc flesh and shells used to treat TB throughout South America; in India, the flesh of marine Turbinella sp. and freshwater Plia globosa is cooked and eaten [114,115]; "snail water" and "snail syrup" prepared from terrestrial Helix sp. were prescribed in Europe since the 1700s (Lemery, 1738 in [91]) and decoctions of numerous marine mollusc species occur in TCM (Tables S1 and S2). Traditional molluscan medicines are also indicated for other infectious respiratory diseases, including influenza, pneumonia, bronchitis, measles, otitis media, and general respiratory tract infections ( Table 2, Tables S1 and S2) and biomedical studies have targeted responsible pathogens (Table 4, Table 5 and Table S3). Two biomedical studies have shown good in vitro activity of molluscan compounds against M. tuberculosisanalogues of kahalalide F [116] from a tropical sea slug, and lobophorins from Streptomyces sp. associated with a gastropod from the Philippines [117]. However, in both cases, effective concentrations exceed toxicity estimates, rendering these compounds unsuitable as therapeutic candidates against M. tuberculosis without modification to reduce toxic side effects (Table 5). Non-tuberculosis mycobacteria (NTM) infection (arising from mycobacteria other than M. tuberculosis) is a clinically severe and rapidly increasing problem worldwide (Table 1) [28]. No reference to this disease was found in traditional or contemporary literature and it should be a subject of further investigation. * Containing at least one, usually several different, mollusc species; one remedy may be used to treat various conditions in which case it has been counted more than once; a remedy is included once if various words/phrases were used in the cited text relating to the same condition. † Not including tuberculosis. ‡ Pulmonary and extrapulmonary. § Included if remedy used for at least one specified respiratory condition.
Asthma represents another universal respiratory disease, albeit of a completely different nature to TB, being non-communicable and a function of inherent immune dysregulation, with both genetic and environmental factors ( Table 1). Records of eight TCMs and 19 other traditional molluscan medicines refer to the treatment of asthma ( Table 2, Tables S1 and S2). Notable ones include tea of toasted cuttlefish bones or octopus arms taken among traditional Brazilian communities [122], soup prepared from the foot of freshwater snail Filopaludina sp. in India, and preparations of mollusc flesh eaten in China [78], India [114], and South America [113] (Table S1).
Most TCMs and other traditional medicines are applied for multiple, possibly related, respiratory conditions ( Table 2, Tables S1 and S2). For example, the TCM "Yan Qiu Bei" derived from the decocted shell of the cowrie Naria erosa (Cypraeidae) is used for extrapulmonary TB, shortness of breath, excessive mucus, and conjunctive congestion [104] (Table S2), while Indian remedies using Turbinella sp. are not only used for TB, but for cough, excessive mucus, sore throat, fever, earache, and asthma as well [114] (Table S1). It must be noted that most of the TCMs and other traditional medicines used for respiratory diseases (Tables S1 and S2) are commonly used for other, non-respiratory conditions (e.g., gynecological problems, gastrointestinal disorders and cardiovascular diseases), which may reflect general anti-inflammatory and anti-angiotensin properties.
There are few references to cancer in general, or respiratory cancer specifically, among traditional molluscan medicines ( Table 2, Tables S1 and S2). Those in this review were included because they are/were also used for at least one other definitively respiratory condition: Crassostrea rhizophorae flesh and shell used in South America [113] for cancer, TB, flu and pneumonia, opercula of the Muricid mollusc Chicoreus virgineus used in Medieval Egypt for tumor and eye/ear diseases [126,131], and the TCM "Mu Li" comprising the flesh and shell of 12 Ostreidae/Grypaeidae sp. for cancer and TB [104] (Table 2, Tables S1 and S2). Descriptions of the preparation and application of each of these medicines is unavailable or ambiguous (Tables S1 and S2), in alignment with Cragg et al. [93,132] who advises that claims for cancer treatments should be viewed with some skepticism because the disease is likely to be poorly defined in traditional medicine. Respiratory cancer, insidious and inconspicuous by nature, may be masked by other symptoms described in the literature (e.g., pneumonia, hemoptysis, shortness of breath, tightness or fullness in chest; Tables S1 and S2). Nonetheless, there exists plenty of biomedical evidence regarding the anticancer activity of molluscan extracts and compounds, some with specificity to respiratory cancers, discussed later in this review (Tables 4 and 6-8).
Among different traditional medicine systems, certain molluscan families, species and body parts are commonly used suggesting shared properties. For example, mucus of terrestrial snails has been widely consumed as a cough remedy with records of use in Africa (Limicolaria aurora, Lanistes ovum) [123], China (Limax sp.) [78], and throughout European history (reviewed by Bonnemain [91]) (Tables S1 and S2). Intertidal periwinkles (Littorinidae) are used in South America [123] and Africa [123] for symptoms of respiratory inflammation (Table S1). Different parts of the cuttlefish Sepia officinalis were used in Ancient Greece [109] and more recently in India [114] and Europe [120], while the remedy "Sepia", manufactured from S. officianalis ink [120] has been part of the Homeopathic Materia Medica since the 1800s [133]. However, this and other homeopathic remedies are typically prescribed at dilutions of 6C or 30C (equivalent to 10 −12 or 10 −60 , respectively). At such high dilutions the presence and bioactivity of the original compounds is conceivably negligible [90].
The shell (and whole body, including shells) is the molluscan part most frequently used in traditional medicines for respiratory diseases ( Table 2, Tables S1 and S2; Figure 1). In TCM, shells are ground into powder or decocted (heated to extract essence) and ingested (Table S2). Ashes of burned mollusc shells or cuttlefish bones, prepared into pills, pastes, and solutions, feature in several traditional respiratory medicines originating in India [114] and Ancient Greece [109]; shells are also used in South American medicine [113,121] and homeopathy [118][119][120]. Only three biomedical studies have investigated shell extracts with relevance to respiratory disease: powdered cowry (Monetaria moneta) shell [134] and chitosan from cuttlefish (S. officianalis) bone [135,136], both which show antimicrobial properties in vitro ( Figure 1; Table 5).
The shell-less body (or "flesh", and "whole animal" including the body) is the second most utilized molluscan part in traditional medicines for respiratory diseases of every nature ( Table 2, Tables S1 and S2; Figure 1). Flesh may be cooked and eaten [109,115] or applied externally [104,109] (Tables S1 and S2). In Ancient Greece, flesh of cephalopods (O. vulgaris, S. officianalis) and the giant trumpet shell (Charonia tritonis) were eaten for conditions of low immunity in addition to infectious or inflammatory respiratory diseases [109] (Table S1). Among Nigerian tribes, the flesh is "punctured to obtain the fluid" ( [123] p. 491). These accounts could be related to circulating hemolymph (equivalent to blood), which represents at least 20% of molluscan body weight [137]. Hemolymph is composed largely of hemocyanin (Hc), which has a range of bioactivities and is among the most potent of immunogens used in respiratory disease-related studies, as discussed later in this review (Tables S3  and S4). Many other bioactive compounds (e.g., AMPs, proteins, peptides, and polyketides) have been derived from molluscan body tissue (Tables 5-8 and Table S6, Figures 2 and 3).

Supporting Evidence for the Bioactivity of Traditional Molluscan Respiratory Medicines
The bioactivity and therapeutic value of traditional molluscan medicines is for the most part speculative. However, there are growing bodies of substantiating evidence by virtue of biomedical studies using derivatives of hemolymph, mucus, body extracts, and specialized glands ( Figure 1, Tables 3-8). European 18-19th century physicians and pharmaceutical texts proclaimed the unsurpassed benefits of helicidine (mucus of the terrestrial snail H. pomatia) for alleviating various respiratory conditions such as whooping cough, TB, influenza, pneumonia, chronic bronchitis, and asthma [91] ( Table S1). In 1953, Quevauviller [138] reviewed the pharmacology of helicidine relating its effectiveness to demonstrated sedative, mucolytic and bacteriolytic activities, and in 1999, Pons et al. [79] established that the broncho-relaxant effect of helicidine in vitro was mediated by the release of prostaglandin E 2 and inhibited by pre-treatment with a cyclooxygenase (COX) inhibitor, indomethacine (Table 6). A double-blind, placebo controlled clinical trial followed in 2001 [80] whereby 30 patients with COPD were treated with daily doses of helicidine (10%) over five days, resulting in a significant reduction in nightly coughing episodes (4.7-5.1 pre-treatment, 2.7-4.9 placebo, 1.3 helicidine group) and duration of coughing periods (Table 8).
Other terrestrial land snails, of the Limax genus, originally appeared in the Chinese Compendium of Materia Medica in 1578 and continue to be used in TCM for respiratory wheeze, phlegm, and pharyngitis [78] ( Table S2). In a recent robust murine model of cigarette-smoke induced COPD undertaken by Liang et al. [78], aqueous body extract of Limax sp. significantly improved pulmonary function and reduced key inflammatory mediators ( Table 7). The effect is attributable to enhancement of peroxisome proliferator-activated receptor-γ (PPAR-γ), which acts to downregulate pro-inflammatory transcription factors and mucin synthesis, and suppression of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) pathways, which activate the production of pro-inflammatory mediators [17], and also play a role in malignant transformation [139], in structural and immune cells of the lung. In other models, systemically administered Limax sp. powder-water suspensions have been shown to inhibit Lewis lung carcinoma growth in mice [111], and reduce the onset of asthma symptoms and production of inflammatory markers in guinea pigs more effectively than the clinical bronchodilator Aminophylline [140] (Table 7).
The flesh and shell of Muricidae sp. occur in eight TCMs [104] and their operculum were ingested as a medicinal oils in Ancient Greece [109], Ancient India [126,131], and Medieval Egypt [126,127] for various infectious and inflammatory respiratory diseases, and cancer ( Table 2, Tables S1 and S2). Considerable attention has been devoted to Tyrian purple (6,6 dibromoindigo and related compounds), a historically important textile dye obtained from the Muricidae family, which was first described in the Historia Naturalis (1669) [141][142][143][144][145]. Natural product research has since ascertained the broad spectrum antibacterial, anti-inflammatory, and anticancer activity of Tyrian purple precursor compounds, minor pigments, and modified derivatives [49,53,75,77,141,[146][147][148]. Extracts of an Australian whelk Dicathais orbita containing these bioactive compounds have shown preliminary antimicrobial activity against respiratory pathogens in vitro [149] and anti-inflammatory activity in a murine model of acute lung inflammation [77] (Table 7). Some bioactive compounds are also detectable in lipophilic extracts of D. orbita operculum [126], with relevance to ancient medicinal oil preparations [127,131] (Table S1). Nonetheless, the only Muricidae sp. that have been the focus of investigations pertaining directly to respiratory disease are D. orbita (brominated compounds) [77] and Rapana venosa (Hcs) [76,150] (Table 7 and Table S3, Figures 3 and 4), while at least 30 different Muricidae sp. are used in traditional respiratory medicines (Tables S1 and S2).

Taxonomic and Geographic Trends
The majority of traditional medicines and biomedical studies are based on extracts and compounds from the Gastropoda and Bivalvia (Table 3), which is unsurprising given that these classes comprise the majority (>90%) of molluscan biodiversity [53]. There are 21 families of Bivalvia used in traditional medicines that have not yet been investigated scientifically. Only one study has investigated the anti-inflammatory activity of Cephalopoda extracts [151], despite their widespread traditional use as anti-inflammatory medicines [109,[120][121][122] (Tables S1 and S2). The remaining four classes are all marine and relatively minor in terms of their ecological diversity and representation in both biomedical and ethnomedical literature (Table 3) [53]. However, the Polyplacophora (chitons) are of research interest, also noted by Benkendorff [53], given their ecological abundance and use in TCM for asthma, TB, and bronchitis (Table S2). Chitons also form important South African traditional medicines, but not for respiratory disease [108]. The Scaphopoda (tusk shells) represent a unique evolutionary avenue within the Mollusca [152]; they are the only class of exclusively infaunal molluscs and could yield interesting chemistries but remain poorly examined to date (Table 3) [152]. The Aplacophora and Monoplacophora mostly occupy the deep-sea making for challenging access and limited utility, although such extreme adaptive radiation in their biology suggests that they are also likely to be chemically unique [53].  Marine  46  17  55  8  3  1  Freshwater  0  3  2  0  0  0  Terrestrial  0  4  5  2  1  1 * Literature based on marine sp. only [104]. † Hc used as a model antigen in in vivo models and clinical trials.
Regional environmental factors have important bearings on the chemical and biological diversity of mollusc species used as traditional medicines in different cultures. A large proportion of traditional medicines included in this review represent Asian (52%), European (24%), and South American (5%) cultures and species endemic to these regions. By comparison, biomedical research efforts have been focused in Europe (34%), North America (28%), and Asia (China and India, 27%). Only one South American species has been studied [153,154] and Australian species are also underrepresented (Table S1). Numerous sources attest to the importance of molluscs as traditional food sources as opposed to traditional medicines in Australia and throughout the Indo-pacific [155,156]. It is possible that molluscs are also used for medicinal purposes but the knowledge has been retained by Indigenous communities or otherwise lost. Accordingly, species from these regions do not appear in our review of traditional medicines, but are nevertheless likely to have some nutraceutical qualities equivalent to molluscs found elsewhere.
Respiratory diseases have changed over time and modern society now faces pathogens and environmental hazards once unimagined [33]. Nonetheless, traditional medicines have not lost their place and continue to provide stimulus for research. Those included in this review incorporate over 300 different mollusc species (Tables S1 and S2), while only 93 species have been investigated in biomedical studies to date (Tables S3-S8). It is estimated that 80% of people in developing countries still rely on traditional medicines as a primary source of healthcare [157] and well-known systems, including TCM, Indian Ayurveda and homeopathy, are expanding in global popularity throughout the developed world, despite a lack of robust evidence for their safety and efficacy [157][158][159]. Further testing is needed to substantiate the use of traditional molluscan medicines and identify bioactive compounds that could serve as APIs and novel drug leads for respiratory disease.

Overview
At least 97 peer-reviewed biomedical articles published mostly within the past two decades investigate the bioactivity of molluscan extracts and compounds with direct relevance to the treatment, prevention or understanding of respiratory disease (Table 4). A small proportion (less than 2%) of the 327 individual extracts/compounds tested in vitro have progressed though in vivo models of respiratory disease to clinical trials (Table 4). Only two have been approved for use although at least 12 have been patented for development toward respiratory disease (Table S5). Fewer than half of studies have used purified compounds (Table S6). Those compounds that have been purified include polyketides (e.g., Figure 2), proteins, glycoproteins (e.g., Figure 3), PUFAs, and brominated indole/isatin derivatives (e.g., Figure 4) ( Table S6).
The in vitro studies included in this review are focused on antimicrobial properties, usually expressed as a minimum inhibitory or bactericidal concentration (MIC or MBC, respectively), representing the minimum concentration of an extract/compound required to inhibit the growth of microbial cells or kill them entirely; and, anticancer properties whereby the indexes IC 50 , EC 50 , GI 50 or CC 50 (50% inhibitory concentration, effective concentration, growth inhibition or cytotoxic concentration, respectively) are used often and interchangeably, representing the concentration of an extract/compound that reduces cell growth in vitro by 50% compared to the untreated control. In vitro studies have used extracts/compounds derived from the hemolymph, sperm, tissue associated with symbiotic bacteria, shell, salivary gland, digestive gland, egg mass, body, and mucus of different mollusc species (representing 56 families) ( Figure 1, Table 3). Further testing and characterization of crude extracts used in many of these studies may lead to the identification of novel bioactive lead compounds, which could become productive drug leads (Tables 5 and 6).
In vivo studies are based on mouse models of respiratory disease (COPD, allergic and acute inflammatory airway disease, lung cancer and respiratory infection) treated with molluscan extracts/compounds. More than half (13) of these models use Hc for its immunogenic properties in vaccine preparations targeting respiratory pathogens (Table S3). Another three extracts and 10 compounds are derived from the body, hemolymph, or hypobranchial gland of different mollusc species with a focus on their anti-inflammatory and anticancer properties (Tables 4 and 7). A separate body of research uses molluscan Hcs as a model antigen in respiratory disease-related immunological studies (Table S4). The focus of these studies is not on the therapeutic value of Hc, rather they provide insight into the bioactivity, safety, and biomedical applications of Hc and functional units.
We identified 11 human clinical trials using purified molluscan compounds as treatments for respiratory disease (Tables 4 and 8). These trials have used keyhole limpet hemocyanin (KLH) as a vaccine adjuvant/conjugate (n = 4) (Table S3), kahalalide F (and derivatives) for lung cancer (n = 3), helicidine for COPD (n = 1), and Lyprinol for asthma (n = 3) ( Table 8). Lyprinol is now FDA approved for use as an alternative anti-inflammatory agent, and helicidine-containing cough medicines have been available over-the-counter in European pharmacies for over 50 years [160]. The overall benefits of other clinically tested molluscan compounds have been marginal (Table 8) such that they are being further developed with structural modifications (Table S5).

Antimicrobial Activity
Research on molluscan antimicrobial chemistry and immunology has been driven mainly by development of the commercial aquaculture industry and microbial disease outbreaks among major cultured mollusc species (e.g., oysters, abalone and mussels [161][162][163][164]). However, the bioactivity of molluscan extracts and compounds is not strictly limited to molluscs and the pathogens that affect them. Antimicrobial susceptibility test suites often include bacterial, viral, and fungal pathogens responsible for common respiratory infections in humans (e.g., [65,73,136], Table 5). The prevalence, intrinsic virulence, and progressive antimicrobial resistance of respiratory pathogens continues to drive research interest and some novel molluscan compounds show promising bioactivity. Even so, most antimicrobial work has only been performed in vitro, and to describe a compound or effect as "promising" based on this data alone is to forget that a disease in a living organism is much more complex; hence, the need for in vivo testing. Pseudomonas aeruginosa is particularly associated with chronic bacterial infections in COPD, cystic fibrosis, and conditions of compromised immunity (Table 1) and its treatment is complex because of recurrence and multi-drug resistance [165,166]. Given the clinical need, P. aeruginosa is commonly included in antibacterial assays (Table 5; 23 studies), although activity against it is generally weak due to the protective lipopolysaccharide outer cell membrane characteristic of this and other Gram-negative bacteria [73,167,168] (Table 5). Reasonable antibacterial activity against P. aeruginosa has been observed using some molluscan extracts and compounds (Table 5).
Examples include: tyriverdin (MIC 0.005 mg/mL) sourced from egg masses of D. orbita [149]; tartrolon E (MIC 0.31 mg/mL) a polyketide isolated from a molluscan gill symbiont [169]; peptides from O. vulgaris (MIC 50-300 µg/mL) [170]; Scutinin A isolated from Australian limpet Scutus antipodes (MIC 33 µM) [171]; 5'-deoxy-5'-methylthio-adenosine (MTA) from a dorid nudibranch [172]; and body extract of Drupella margariticola (MIC 0.07 mg/mL) [173] (Table 5, Figures 2 and 4). Preliminary assays using mucus of H. aspera [174] and crude extract of Babylonia spirata [175], show antimicrobial activity although weaker than antibiotic controls (Table 5). Notwithstanding, crude extracts would sensibly become more effective if the active compound/s were concentrated by purification. Hence, extracts that show weak activity should not necessarily be overlooked in case the active factor is a minor component. The capacity to form biofilms (structured communities of bacteria encapsulated within an extracellular polymeric matrix) confers some bacteria, including P. aeruginosa, an enormous advantage in establishing and maintaining infections in the respiratory tract [177]. Of interest therefore are studies by Gasu et al. [178,179] using peptide extract of Olivancillaria hiatula establishing a relatively low MIC of 0.039 mg/mL against P. aeruginosa and 50% inhibition of biofilm formation at the same concentration, though 2.5 mg/mL was required to degrade pre-formed biofilm (Table 5). Further, the peptide extract reduced the expression of virulence factors (pyocyanin, pyoverdine, and protease) by >50% at 0.0195 mg/mL and acted synergistically with standard antibiotics ciprofloxacin and cefotaxime (up to 100% reduction in MICs) ( Table 5). As well, Maselli et al. [170] recently reported antibiofilm activity (60% eradication at 80 μM) among peptides derived from the suckers of O vulgaris. Preliminary evidence indicates that Hcs from marine crustaceans can also inhibit biofilm formation (at 0.1 mg/mL) [180][181][182] and molluscan Hcs could function similarly.
Community-acquired pneumonia is the leading cause of death from infection worldwide, particularly in infants, and is typically caused by Streptococcus pneumoniae [2,183]. Bacteriostatic activity against S. pneumoniae has been observed using crude body extracts of D. margariticola (MIC 0.07 mg/mL) [184], Babylonia spirata [175], and several Cephalopods at high concentrations [185]. Other molluscan extracts/compounds (e.g., Helicidae sp. Hcs [73] and Muricidae sp. brominated compounds [149]) showing activity against Gram-positive bacteria could be investigated targeting this pathogen.
Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of troublesome healthcare-acquired infections, including pneumonia [186]. Bacteriostatic effects on MRSA have been observed using compounds isolated from molluscan bacterial symbionts [169]. The recent work of Miller et al. [176] using 7,8-dideoxygriseorhodin C (DC) is notable: MIC values for DC (0.08-0.12 The capacity to form biofilms (structured communities of bacteria encapsulated within an extracellular polymeric matrix) confers some bacteria, including P. aeruginosa, an enormous advantage in establishing and maintaining infections in the respiratory tract [177]. Of interest therefore are studies by Gasu et al. [178,179] using peptide extract of Olivancillaria hiatula establishing a relatively low MIC of 0.039 mg/mL against P. aeruginosa and 50% inhibition of biofilm formation at the same concentration, though 2.5 mg/mL was required to degrade pre-formed biofilm (Table 5). Further, the peptide extract reduced the expression of virulence factors (pyocyanin, pyoverdine, and protease) by >50% at 0.0195 mg/mL and acted synergistically with standard antibiotics ciprofloxacin and cefotaxime (up to 100% reduction in MICs) ( Table 5). As well, Maselli et al. [170] recently reported antibiofilm activity (60% eradication at 80 µM) among peptides derived from the suckers of O vulgaris. Preliminary evidence indicates that Hcs from marine crustaceans can also inhibit biofilm formation (at 0.1 mg/mL) [180][181][182] and molluscan Hcs could function similarly.
Community-acquired pneumonia is the leading cause of death from infection worldwide, particularly in infants, and is typically caused by Streptococcus pneumoniae [2,183]. Bacteriostatic activity against S. pneumoniae has been observed using crude body extracts of D. margariticola (MIC 0.07 mg/mL) [184], Babylonia spirata [175], and several Cephalopods at high concentrations [185]. Other molluscan extracts/compounds (e.g., Helicidae sp. Hcs [73] and Muricidae sp. brominated compounds [149]) showing activity against Gram-positive bacteria could be investigated targeting this pathogen.
Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of troublesome healthcare-acquired infections, including pneumonia [186]. Bacteriostatic effects on MRSA have been observed using compounds isolated from molluscan bacterial symbionts [169]. The recent work of Miller et al. [176] using 7,8-dideoxygriseorhodin C (DC) is notable: MIC values for DC (0.08-0.12 µg/mL) were lower than the control antibiotic oxacillin (1.59-6.24 µg/mL), and both the number of colony-forming units and MICs for DC and oxacillin were reduced >100 fold (i.e., synergistic effects) when present in combination (Table 5, Figure 2). Extracts from the sperm of the Mediterranean mussel (Mytilus galloprovincialis) have displayed bactericidal activity against clinical S. aureus strains, as well as negligible toxicity and resistance to acid digestion (i.e., suitable for oral administration) [187]. Wei et al. [188] prepared two recombinant proteins (rSgSABL-1 and -2) from lectins of the razor clam Solen grandis, which exhibited strong binding affinity to S. aureus peptidoglycan, increased phagocytic and encapsulation activity in vitro, and generated reactive antibodies in vivo (Tables 5 and 7). Aside from Wei et al. [188], the antimicrobial activity of molluscan extracts/compounds against respiratory pathogens has not been tested in animals or humans aside from the inclusion of Hcs in vaccine preparations (Table S3).
Terrestrial mollusc mucus is a mixture of proteoglycans, glycoprotein enzymes, AMPs, and other minor constituents in~90% water [189]. Preliminary antibacterial results from disc diffusion assays using some respiratory pathogens are presented by Cilia and Fratini [189], but statistically meaningful data are yet to be derived. de Toledo-Piza et al. (2016) demonstrated impressive anti-viral activity with concentrated mucus from the terrestrial shell-less mollusc Phyllocaulis boraceiensis whereby host cell pre-treatment reduced Influenza A (H1N1) and measles virus-induced cytopathic effects by up to 80% with no host cell cytotoxicity [153,154]. Further studies should incorporate bioassay guided fractionation of concentrated mucus extracts to isolate and identify the active factors.
Numerous published reviews discuss Hcs and AMPs as effectors of invertebrate immunity and their potential as antibiotic leads [102,[190][191][192][193][194][195]. Regardless of their biological origin, all AMPs are small in size, often cleaved from Hcs, with either a cyclic (encompassing amphipathic surfaces) or linear (containing di-sulfide bridges) structure and broad-spectrum antimicrobial activity [164,193] (Figure 3). Molluscan Hcs and AMPs show some inhibition of respiratory bacteria (mostly Gram-positive) and fungi [73,167,168] (Table 5). Although, they may hold even greater potential as antivirals. The review by Dang at al. [56] presents evidence for antiviral effects of molluscan Hcs and AMPs against a range of human viruses including herpes simplex virus, Epstein-Barr virus (double-strand, lipid-enveloped DNA), simian rotavirus (double-strand, non-enveloped RNA) and poliovirus (single-strand, non-enveloped RNA) [56]. Regarding respiratory disease, virucidal activity has been shown using oyster hemolymph tested against human adenovirus (AdV-5) (EC 50 0.05-0.09 mg/mL, cytotoxic at 0.19-0.36 mg/mL) [196] and with Hc subunit c (RvH-c) from R. venosa, which inhibited the cytopathic effects of respiratory syncytial virus (by 71.4% at 1 mg/mL) and indicated that some Hc structural units may be more bioactive than others [197] (Table 5, Figure 3). Nonetheless, negligible toxicity toward healthy eukaryotic cells and low potential for the development of resistance [164,198] are clear advantages for the inclusion of Hcs and AMPs in various antimicrobial and anticancer preparations. cytotoxic at 0.19-0.36 mg/mL) [196] and with Hc subunit c (RvH-c) from R. venosa, which inhibited the cytopathic effects of respiratory syncytial virus (by 71.4% at 1 mg/mL) and indicated that some Hc structural units may be more bioactive than others [197] (Table 5, Figure 3). Nonetheless, negligible toxicity toward healthy eukaryotic cells and low potential for the development of resistance [164,198] are clear advantages for the inclusion of Hcs and AMPs in various antimicrobial and anticancer preparations.  [76,197,199].
Molluscs provide good bioavailable sources of elemental zinc [200]. Zinc was recently shown to inhibit the replication of respiratory viruses (including influenza and COVID-19), reduce host cell infection, improve immune function, and act synergistically with standard antiviral therapies [201][202][203]. Antiviral factors isolated from molluscs could therefore be combined with zinc for improved activity. As well, extracts from whole molluscs, bodies, and shells used in traditional medicines likely contain zinc, which may be in part responsible for some derived benefits (Tables S1 and S2).

Anti-Inflammatory Activity
Scientific and epidemiological evidence suggest a beneficial relationship between dietary PUFA consumption and prevention or alleviation of chronic diseases including asthma, allergic airway disease, cardiovascular disease, and cancer [204][205][206]. Lyprinol™ is a patented extract of the New Zealand green lipped mussel (Perna canaliculus) known to inhibit 5 -lipoxygenase and COX pathways responsible for the production of inflammatory eicosanoids, among other less well-characterised involvement in the production of resolvins and protectins [207]. In a murine model of allergic airway disease, Wood et al. [208] found that daily gavage with 100 µL Lyprinol™ significantly reduced eosinophil influx, mucus hypersecretion, and airway hyperresponsiveness (Table 7). Indeed, different forms of marine oils have different bioactivities since they contain different types and amounts of PUFAs as well as a variety of lipid mediators [207]. Lyprinol™ contains a mixture of PUFAs (including eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA] and other n-3 PUFAs), triglycerides, sterol esters, sterols, and polar lipids combined with olive oil and vitamin E, and these multiple nutritional components may contribute to the superior effectiveness of the supplement over fish oil in animal models [208] ( Table 7) and clinical trials (e.g., [209,210] [ Table 8] compared with [211]).
Ahmad et al. [212] first established evidence for the anti-inflammatory activity of hypobranchial gland (HBG) extract and 6-bromoisatin (among other related compounds) from D. orbita based on the in vitro inhibition of pro-inflammatory mediators (NO, TNF-α and NFκB) in mouse cell lines (Figure 4). In this study, effective, non-toxic concentrations were <50 µg/mL and the purified mono-brominated indole and isatin compounds (i.e., 6-bromoisatin) were more bioactive than their non-brominated counterparts and crude HBG extract [212]. Ahmad et al. [77] was led to investigate whether these anti-inflammatory effects could be replicated in a model of acute lung inflammation, in which HBG extract and 6-bromoisatin significantly inhibited the inflammatory pathway (TNF-α and IL-1β, and neutrophil infiltration) and prevented associated lung tissue damage (Table 7, Figure 4). No mortality, ill-health, liver toxicity, or gastro-intestinal damage was observed in this model, nor others (non-respiratory) using the same extracts/compounds administered over 2-14 weeks (0.05-0.1 mg/g) [48,77,[213][214][215]. Evidently, brominated compounds from D. orbita, and other Muricidae sp., could prove especially valuable for their anti-inflammatory activity and other properties relevant to respiratory disease.
triglycerides, sterol esters, sterols, and polar lipids combined with olive oil and vitamin E, and these multiple nutritional components may contribute to the superior effectiveness of the supplement over fish oil in animal models [208] (Table 7) and clinical trials (e.g., [209,210] [ Table 8] compared with [211]).
Ahmad et al. [212] first established evidence for the anti-inflammatory activity of hypobranchial gland (HBG) extract and 6-bromoisatin (among other related compounds) from D. orbita based on the in vitro inhibition of pro-inflammatory mediators (NO, TNF-α and NFκB) in mouse cell lines ( Figure  4). In this study, effective, non-toxic concentrations were <50 μg/mL and the purified monobrominated indole and isatin compounds (i.e., 6-bromoisatin) were more bioactive than their nonbrominated counterparts and crude HBG extract [212]. Ahmad et al. [77] was led to investigate whether these anti-inflammatory effects could be replicated in a model of acute lung inflammation, in which HBG extract and 6-bromoisatin significantly inhibited the inflammatory pathway (TNF-α and IL-1β, and neutrophil infiltration) and prevented associated lung tissue damage (Table 7, Figure  4). No mortality, ill-health, liver toxicity, or gastro-intestinal damage was observed in this model, nor others (non-respiratory) using the same extracts/compounds administered over 2-14 weeks (0.05-0.1 mg/g) [48,77,[213][214][215]. Evidently, brominated compounds from D. orbita, and other Muricidae sp., could prove especially valuable for their anti-inflammatory activity and other properties relevant to respiratory disease.  Very few investigations of molluscan extracts and compounds are specific to inflammatory respiratory diseases, regardless of all the traditional uses of molluscs relevant to symptoms of inflammation ( Table 2, Tables S1 and S2; [49]) and the prevalence of these conditions (e.g., COPD, asthma, ARDS; Table 1). Those extracts/compounds that have been tested specifically for inflammatory respiratory conditions include: helicidine, studied in vitro with relevance to asthma [79] (Table 5) and in a clinical trial for COPD [80]; Limax sp. extract, studied twice in vivo for COPD [78] and asthma [140]; muricid sp. brominated compounds, studied in vivo for acute lung inflammation [77]; and Lyprinol TM , used in three clinical trials for asthma [207,209,210] (Tables 7 and 8). Notwithstanding, there is significant work dedicated to the general anti-inflammatory activity of molluscan compounds(recently reviewed by Ahmad et al. [49]).

Anticancer Activity
The A549 (human alveolar carcinoma) lung cancer cell line is often used to investigate anticancer activity in vitro, enabling comparison between studies (Table 6). Significant inhibition of A549 proliferation has been shown using a recombinant tumor necrosis factor from the Pacific oyster (200 ng/mL) [247], crude methanol extracts of an intertidal snail Euchelus asper (40% inhibition at 10 µg/mL) [248], and lipid extract from a squid digestive gland (CC 50 260 µg/mL) [249] (Table 6). Liu et al. [250] tested extracts of Ampullarriidae, a family of freshwater snails used in TCM for epilepsy and stomach ache, reporting 31-57% inhibition of A549 between 20-200 µg/mL, and Zhang et al. [251] reports 73-96% inhibition using ethanol extracts of eight Chinese mollusc species, some of which are used in TCM [104]. However, these studies are less robust than others; only three studies included in Table 6 report cytotoxicity against human/animal-derived healthy cell lines, which is critical to ensure specificity [249,252,253] (Table 6).
In assays using a novel peptide (termed Mere15) from the Asiatic clam Meretrix meretrix (Veneridae), Wang et al. [252] found that A549 was more susceptible (IC 50 31.80 µg/mL) than other cancer (breast, colorectal, liver, pancreas; IC 50 43.5-57.4 µg/mL) and healthy (IC 50 123.1-149.5 µg/mL) cell lines ( Table 6). These results prompted the use of A549 in a mouse xenograft model, whereby 50 mg/kg Mere15 administered by subcutaneous injection inhibited tumor growth by 69%, an effect greater than (although statistically indifferent to) the control chemotherapeutic cyclophosphamide (53% inhibition at the same concentration) [252] (Table 7). The Veneridae are valued in TCM with 42 different species used in 11 remedies for inflammatory respiratory diseases (e.g., asthma, cough, tuberculosis, tracheitis), but not cancer [104] (Table S2); Veneridae sp. are also used as medicines in South America for asthma and influenza [113] (Table S1). Anti-inflammatory activity of extracts and compounds from this family is a topic certainly worth pursuing, in conjunction with anticancer work.
The best available evidence for molluscan anticancer compounds to date comes from kahalalide F (KF) (a cyclic depsipeptide), dolastatin-10 (a linear pentamer with four unique amino acids), and structurally related analogues, which have progressed from in vitro through in vivo studies into Phase I and II clinical trials for respiratory, and other, cancers (Tables 6-8, Figure 5). The strong activity of dolastatin 10 is illustrated by Kalemkerian et al. [254], who report low IC 50 s (0.03-0.184 nM) against a panel of lung cancer cell lines ( Table 6) and found that two doses of 4.5 mg/kg dolastatin 10 could completely inhibited tumor formation, or significantly reduce the size of established tumors (1635 mg control vs. 44 mg dolastatin-10) and improve survival in a H446 (small cell lung cancer) xenograft model (Table 7). Once a lead compound is identified, a research team may synthetically explore and alter the structure in order to maintain its favourable properties while improving on deficiencies, as did Kobayashi et al. [255] who found that TZT-1027, a synthetically modified dolastatin-10 derivative designed for enhanced bioactivity and lower toxicity, resulted in 84-98% regression of pre-established lung cancer xenograft tumors with just one or two treatments at 1-2 mg/kg (Table 7, Figure 5). GI 50 /IC 50 values for KF against A549 range from 0.135-1 µM (0.20-1.48 µg/mL) [253,256,257], which are far lower than cytotoxic concentrations (>4.76 µg/mL [253]) ( Table 6). A549 appears more sensitive to KF than other cancer types in vitro (e.g., colon, leukemia, melanoma, ovarian, prostate; [253,257]), although other respiratory cell lines are more resistant (e.g., H460 [256], HOP62 [258]). Three clinical trials involving KF and elisidepsin (a KF derivative; Figure 5) have shown their capacity to stabilize lung cancer (by induction of apoptosis, inhibition of microtubule formation and blockade of cellular growth pathways, as opposed to immunomodulatory mechanisms) at maximum tolerable doses of around 6.5 mg/m 2 [259][260][261][262] (Table 8). Interestingly, elisidepsin has been shown to act synergistically with paclitaxel, cisplatin, and gemcitabine against lung cancer cell lines in vitro [258] and with Erlotinib in vivo, significantly enhancing the survival of mice with A549 tumors (elisidepsin 54 d, erlotinib 39 d, combination >150 d, control 23 d) [263] (Tables 6-8). Such combinations of molluscan compounds with approved drugs may help to overcome chemotherapy resistance. Myelosuppression and elevated transaminase levels are dose-limiting drawbacks of KF and dolastatin compounds in vivo, as per many chemotherapeutic agents [254,260,264,265]. xenograft model (Table 7). Once a lead compound is identified, a research team may synthetically explore and alter the structure in order to maintain its favourable properties while improving on deficiencies, as did Kobayashi et al. [255] who found that TZT-1027, a synthetically modified dolastatin-10 derivative designed for enhanced bioactivity and lower toxicity, resulted in 84-98% regression of pre-established lung cancer xenograft tumors with just one or two treatments at 1-2 mg/kg (Table 7, Figure 5).  (Table 6). A549 appears more sensitive to KF than other cancer types in vitro (e.g., colon, leukemia, melanoma, ovarian, prostate; [253,257]), although other respiratory cell lines are more resistant (e.g., H460 [256], HOP62 [258]). Three clinical trials involving KF and elisidepsin (a KF derivative; Figure 5) have shown their capacity to stabilize lung cancer (by induction of apoptosis, inhibition of microtubule formation and blockade of cellular growth pathways, as opposed to immunomodulatory mechanisms) at maximum tolerable doses of around 6.5 mg/m 2 [259][260][261][262] (Table 8). Interestingly, elisidepsin has been shown to act synergistically with paclitaxel, cisplatin, and gemcitabine against lung cancer cell lines in vitro [258] and with Erlotinib in vivo, significantly enhancing the survival of mice with A549 tumors (elisidepsin Original sources of KF and dolastatin-10, the tropical ornate leaf sea slug, Elisia ornata (Plakobranchidae) and the sea hare, Dolabella auricularia (Aplysiidae) respectively, are rarely used in traditional medicine, with the exception of an unknown Aplysiidae sp. body part used in Middle Eastern medicine (for cough, dyspnea, and hemoptysis) and egg masses used in one TCM (for cough, TB and dyspnea) (Tables S1 and S2). Aplysiidae sp. egg masses contain a range of bioactive compounds, but not dolastatins, [266] and there is evidence for acute liver damage upon ingestion [267]. The bias of natural product cancer research toward heterobranch gastropods, in which the shell is often reduced or entirely absent, is not justified by the potential for toxic (anti-predatory) compounds and the fact that traditional molluscan medicines are largely derived from shelled species (Tables S1 and S2, Figure 1) [53].
The lung is the preferred site for metastases of common cancers originating elsewhere (e.g., breast, colon, prostate and bladder) in up to 52% of cases [268] and such metastases are the single most negative factor in the cancer prognosis [269,270]. Gesheva et al. [76] measured lung metastases in a murine model of colon cancer, whereby direct solid C-26 (colon) tumor injection with Hcs from R. venosa (RvH; Figure 3) or Helix pomatia (HpH) resulted in significant reductions in tumor size, high anti-C-26 antibody levels, marked reductions in surface lung metastases and up to 30% increased survival after >13 weeks ( Table 7). The effectiveness of RvH was dependent on priming before tumor inoculation, yet HpH was effective even without it [76], suggesting that the mechanism of action is related to modulation of cellular immunity (i.e., natural killer cells, antigen presenting cells, and specific CD8 + and CD4 + T cells) by Hc and subsequently, better recognition and elimination of abnormal cells [271,272].
Synergeneic (or allograft) models involve the transplant of immunologically compatible cancer cells into immunocompetent mice [273]. Because of these qualities, these models are useful to study interactions between tumors, functional host immune systems and treatments, and eliminate the potential for tumor rejection that exists in xenograft models unless mice are first immune compromised. The only reproducible syngeneic model for lung cancer to date is the Lewis lung carcinoma model [273], as used by Gomes et al. [274], who measured the formation of lung metastases in mice treated with a heparin-like glycan from Nodipecten nodusus, a bivalve mollusc found along the north Atlantic coast of America. In this study, the size and number of metastatic lung foci were significantly reduced, from ten to one per lung (PBS control and molluscan heparin treatment groups, respectively) [274] ( Table 7). The in vivo and in vitro components of the study by Gomes et al. [274] demonstrated that molluscan heparin inhibits P-selectin interaction with colon carcinoma (LS180) and reduces inflammatory cell recruitment, platelet-tumor cell complex formation, and heparinase enzymatic activity, thereby attenuating metastases, although not to an extent beyond its mammalian counterpart. Heparin analogues from other mollusc species that possess strong anticoagulant activity [275][276][277][278]] are yet to be tested as chemotherapeutics.
Abundant literature has been published on the anticancer activity of brominated indole and isatin derivatives from muricid molluscs in vitro [75,147,251,[279][280][281][282][283][284][285] and in in vivo models of cancer treatment [285,286] and prevention [48,214,287,288] (Figure 4). Zhang et al. [251] present the only evidence for Muricidae sp. extracts against respiratory cancer, indicating that they can inhibit A549 proliferation and regulate adaptive immunity in vitro, but further analysis is required to determine significance and identify which, if any, extracts are productive to pursue. The recent review by Ciavatta et al. [66] covers other potentially promising mollusc-derived anticancer agents, including terpenes, steroids, peptides, polyketides, and nitrogenous compounds, currently untested against respiratory cancers.

Molluscan Hemocyanins as Therapeutic Adjuvants and Model Antigens
When inoculated into mammals, molluscan Hcs have remarkable immunogenic effects: they generate a strong cellular reaction, promote high antibody levels and Th 1 /Th 2 cytokine release, and prime antitumor CD8 + and CD4 + T cells [272] (Figure 3). Because of these versatile properties along with their large size (3.3-13.5 MDa), Hcs are regularly used as vaccine adjuvants and hapten carriers (subunit conjugates) [103]. As biomolecules, they are also considered safer than synthetic adjuvants (e.g., aluminium derivatives) [83]. Keyhole limpet hemocyanin (KLH) from the marine gastropod Megathura crenulata (Fissurellidae) is the most extensively used Hc, and is commercially available to biomedical researchers for vaccine development and immunological studies [103] (Tables S3 and S4). KLH has also been prescribed as an adjuvant in superficial bladder carcinoma therapy for over 30 years, and has shown potential in the treatment of other epithelial-derived adenocarcinomas, including of the lungs and respiratory tract [103].
There also exists a large body of research using molluscan Hcs (again, mostly KLH) as a model protein antigen, which has contributed to the assessment and understanding of immune responses involved in respiratory diseases (Table S4). For example, atopic asthmatics have been shown to produce more serum anti-Hc IgG4 [314] and IgE [30] than normal individuals, indicative of an increased/overactive Th 2 response, while administration of molluscan Hcs to patients with respiratory cancer has revealed the immunosuppressive effects of chemotherapy and the disease itself [315,316] (Table S4). Sensitisation to KLH has been found to have an effect that is more-so protective than allergenic [317,318], but serum IL-4 and anti-KLH IgG4 and IgE increase in the presence of diesel exhaust particles [319], demonstrating the promotion effect of environmental pollutants on allergic airway diseases (Table S4). KLH has an excellent clinical safety profile [103] with no adverse events related to the use of this, or other Hcs, in any study reviewed for this paper (Table S3).
The versatile biomedical applications of KLH have led to increasing commercial demand and interest in obtaining novel Hcs with better/different immunogenicities [313]. Gesheva et al. [83,150] carried out two models whereby mice were immunised with influenza hemagglutinin subunit with adjuvant Hcs from H. pomatia (HpH) and R. venosa (RvH; Figure 3): although antibody and cytokine profiles varied, HpH produced immunogenic effects comparable to alum (AlOH 2 ); RvH was comparable to KLH and Complete Freund's Adjuvant, and ultimately superior to KLH in terms of spleenocyte cytotoxicity to virus-infected cells three-months post-immunisation (Table S3, Figure 2). HpH has also been used effectively as a model antigen in studies of asthma [30,320] and bronchial carcinoma [315] ( Table S4). Hc from the Chilean abalone (Concholepas concholepas) is known to have immunogenic properties equal to or better than KLH; it is also commercially available and currently used in a clinical trial as an adjuvant in a prostate cancer vaccine [272].

Sustainable Supply and Traditional Knowledge Considerations
A critical question concerning natural product research is the technical and economic feasibility of obtaining large quantities of a given compound in a consistent and ecologically sound manner [321]. The ecology and life-history of a given species should be well-understood prior to intensive study and opportunities for non-lethal harvest should be sought where possible. Chemical synthesis of bioactive compounds is preferred for pharmaceutical supply due to the relative ease of quantification and purity assessment of active factors, although this is generally not suitable for nutraceuticals which imply a natural origin [141]. Sustainable production, with respect to both compound sources and other materials used in experimental processes, will be essential for the registration of new molluscan medicines by the FDA, Therapeutic Goods Administration, and counterpart authorities in developed countries.
The identification and collection of samples based on traditional knowledge adds a layer of legal complexity to the drug discovery process. For many years, it has been recognized that countries and indigenous groups have the right to take control of their biological property, and that the knowledge forming the basis of their traditional medical practices could be protected [322,323]. This is a contentious topic and opinions on its resolution vary widely. Nonetheless, as summarized well by Cordell & Colvard [323], acknowledging and compensating indigenous groups for their knowledge and for providing access to the local environment is a reasonable expectation for those who hold the resources, as well as those who seek them.

•
This review highlights that there is a paucity of research on the bioactivity of molluscan extracts and compounds, considering the high diversity of species in this phylum and their merit as traditional medicines. Here, we have demonstrated the links between the anti-inflammatory, antimicrobial, anticancer, and immunomodulatory activity of molluscan extracts and compounds and their therapeutic potential in the prevention and treatment of respiratory diseases. • At least 100 traditional medicines incorporating over 300 species of Mollusca have been used to treat respiratory diseases for thousands of years. Most of these are yet to receive research attention, and those few that have shown interesting bioactivities that validate some applications.
There is a continued need to develop an evidence base toward the integration of quality-controlled traditional medicines.

•
We identified particular incentive for biomedical research that elucidates anti-inflammatory factors from mollusc species/families comprising traditional medicines as there is likely to be some chemical basis consistent with their extensive use for alleviating inflammatory symptoms. Shell extracts are widely used but understudied and worthy of further investigation, as are certain taxonomic classes and families used in traditional medicines. The Polyplacophora and Scaphopoda, a wider range of Bivalvia including Veneridae, and shelled Gastropoda including Muricidae are of interest. Respiratory disease-focused ethnomedical studies would also be useful.

•
Based on biomedical data, we expect that studies using molluscan compounds isolated from specialized glands, reproductive organs, microbial symbionts, and hemolymph would prove worthwhile.

•
The exploration of novel molluscan Hcs holds good potential for the discovery of new antiviral and immunomodulatory agents, and therapeutic alternatives to KLH. Hcs could be tested in combination with other antiviral factors (e.g., zinc) from the same mollusc. Snail mucus should also be further investigated for anti-spasmodic, anti-inflammatory and anti-viral activities.

•
Molluscan compounds may be valuable in the treatment of biofilm-associated respiratory infection and improve the efficacy of antibiotics. Further biofilm inhibition/disruption studies are needed and should be inclusive of S. pneumoniae and P. aeruginosa.

•
Derivatives of KF and dolastatin 10 are the most potent molluscan anticancer compounds and of continued interest. Several compounds show anticancer activity and need to be tested against respiratory cancers (e.g., brominated indole/isatin derivatives; others in [66] and structural modifications). In vivo models of various cancer types should include measures of lung metastases and cytotoxicity to healthy cells.

•
Compounds with bioactivities relevant to a range of respiratory diseases (e.g., anti-inflammatory activity) should be further explored, as well as combinations of compounds (molluscan-molluscan and molluscan-standard agents) to improve treatment efficacy for a single disease and address issues of chemotherapeutic and antimicrobial resistance.

•
Overall, there is a need for more targeted research based on specific hypotheses related to respiratory disease using extracts and compounds derived from molluscs, with consideration to the sustainability of supply and attribution of traditional knowledge.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/18/11/570/s1, Table S1: Traditional medicinal uses of molluscs for respiratory related conditions used in ancient and modern cultures around the world appearing in ethnomedical texts and peer-reviewed articles. Table S2: Traditional Chinese medicines (TCMs) derived from molluscs relevant to the treatment of respiratory disease listed in the Chinese Marine Materia Medica. Table S3: Human clinical trials and in vivo animal models using molluscan hemocyanins as vaccine adjuvants/conjugates for respiratory disease. Table S4: In vivo animal models and human studies using molluscan hemocyanins as a model antigen for research investigating respiratory diseases. Table S5: Selected patents relating to pharmaceutical compounds derived from molluscs for the treatment of respiratory disease. Table S6: Classes of purified compounds tested against respiratory pathogens, cancers and inflammatory diseases.

Funding: This research was funded by the Australian Government Research Training Program and Southern
Cross University School of Environment, Science and Engineering.